Poly(ethylene glycol)-modified silk fibroin membrane as a carrier for limbal epithelial stem cell transplantation in a rabbit LSCD model.
Poly(ethylene glycol)-modified silk fibroin membrane as a carrier for limbal epithelial stem cell transplantation in a rabbit LSCD model.
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DOI：
10.1186/s13287-017-0707-y
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发表时间：
2017-11-07
影响因子：
7.5
通讯作者：
Xu H
中科院分区：
文献类型：
--
作者：
Li Y;Yang Y;Yang L;Zeng Y;Gao X;Xu H
角膜缘上皮干细胞（Limbal epithelial stem cells, LESCs）在角膜上皮稳态和再生中起重要作用，角膜缘损伤会导致角膜缘干细胞缺乏症（Limbal stem cell deficiency， LSCD），结膜炎甚至视力障碍。培养的LESCs已被用于眼表重建，丝素（SF）膜已显示出作为LESCs培养底物的潜力。LESCs的培养方法和载体都会影响LESCs移植后的预后。采用组织外植体培养法、单细胞悬浮培养法和细胞团培养法培养兔LESCs。检测p63α和/或abcb5阳性的LESC、分化的角膜上皮细胞（CK12染色）和角膜紧密连接形成（cladin -1染色）的比例，以选择最适用的LESC培养。采用400-Da聚乙二醇（PEG）对SF膜进行了改性。通过免疫荧光染色和流式细胞术检测聚乙二醇修饰的SF膜上培养的LESCs的干细胞特征和正常角膜分化。将聚乙二醇修饰的SF膜（LESC/SF移植物）和聚乙二醇修饰的SF膜（SF移植物）培养的LESCs移植到完全LSCD的兔角膜上。移植后观察新生血管、角膜上皮缺损和角膜清晰度。并在移植后4个月量化角膜上皮厚度、间质厚度和ck12阳性角膜上皮面积百分比。组织外植体和单细胞悬浮培养比细胞团培养收获更多p63α和/或abcb5阳性的LESCs，产生更多ck12阳性的角膜上皮细胞，形成更多的角膜紧密连接。制备的聚乙二醇修饰的SF膜透明、柔韧、坚固，足以进行手术操作。在聚乙二醇修饰的SF膜上培养的LESCs保持了干细胞的特征和正常的角膜分化。在兔全LSCD模型中，LESC/SF移植抑制新生血管，修复角膜上皮缺损。此外，LESC/SF移植物重新填充角膜缘，增加角膜上皮厚度、间质厚度和ck12阳性角膜上皮面积百分比。组织外植体和单细胞悬浮培养的LESCs更适用于眼表重建。LESC/SF移植修复角膜上皮缺损，逆转LSCD， peg修饰的SF膜适合作为LESC移植的载体。本文的在线版本（doi:10.1186/s13287-017-0707-y）包含补充材料，仅供授权用户使用。
Limbal epithelial stem cells (LESCs) play important roles in corneal epithelial homeostasis and regeneration, and damage to the limbus will lead to limbal stem cell deficiency (LSCD), with conjunctivalization and even visual impairment. Cultured LESCs have been used for ocular surface reconstruction, and silk fibroin (SF) membranes have shown potential as a substrate for LESC cultivation. Both culture methods and the carriers of LESCs affect outcomes following LESC transplantation. Rabbit LESCs were cultured from tissue explant, single cell-suspension, and cell cluster culture methods. Ratios of p63α and/or ABCB5-positive LESCs, differentiated corneal epithelial cells (CK12 staining), and corneal tight junction formation (Claudin-1 staining) were examined to choose the most applicable LESC cultures. SF membranes were prepared and modified by 400-Da poly(ethylene glycol) (PEG). The characteristics of stem cells and normal corneal differentiation of LESCs cultured on PEG-modified SF membranes were further examined by immunofluorescence staining and flow cytometric analysis. LESCs cultured on PEG-modified SF membranes (LESC/SF grafts) and PEG-modified SF membranes (SF grafts) were transplanted onto rabbit corneas with total LSCD. New blood vessels, corneal epithelial defects, and cornea clarity were examined after transplantation. Furthermore, corneal epithelial thickness, stromal thickness, and the percentage area of CK12-positive corneal epithelium were quantified 4 months after transplantation. Tissue explant and single cell-suspension cultures harvested more p63α and/or ABCB5-positive LESCs, generated more CK12-positive corneal epithelial cells, and formed more corneal tight junctions than cell cluster cultures. Prepared PEG-modified SF membranes were transparent, flexible, and sturdy enough for surgical manipulation. LESCs cultured on PEG-modified SF membranes maintained characteristics of stem cells and normal corneal differentiation. LESC/SF grafts inhibited new blood vessels and rescued corneal epithelial defects in the rabbit total LSCD model. In addition, LESC/SF grafts repopulated the limbus and increased corneal epithelial thickness, stromal thickness, and the area percentage of CK12-positive corneal epithelium. LESCs from tissue explant and single cell-suspension cultures were more applicable corneal epithelial cells for ocular surface reconstruction. LESC/SF grafts repaired corneal epithelial defects and reversed LSCD, and PEG-modified SF membranes were suitable to be a carrier for LESC transplantation. The online version of this article (doi:10.1186/s13287-017-0707-y) contains supplementary material, which is available to authorized users.
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影响因子：
14
作者：
Lawrence BD;Marchant JK;Pindrus MA;Omenetto FG;Kaplan DL
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