Hu Xiao-jie, Wang Min, Chai Gang, Zhang Yan, Li Wei-guo, Liu Wei, Cao Yi-lin
Department Plastic Surgery, the 9th People's Hospital, Shanghai Second Medical University, Shanghai Tissue Engineering Research Center, Shanghai 200011, China.
Zhonghua Yan Ke Za Zhi. 2004 Aug;40(8):517-21.
Reconstruct corneal stroma by tissue engineering.
Primary corneal stromal cells were isolated from newborn rabbit cornea. When the cultured cells reaching confluence, the stromal cells were mixed with polyglycolic acid (PGA) to form a cell-scaffold construct. After 1 week in vitro culture, the constructs were implanted into mother rabbit corneal stroma. Parts of corneal stromal cells were transfected with GFP gene as mark of transplanted cells. Tissues were harvested at 8 weeks for transmission electron microscopy (TEM), histology and Western blot evaluation. In control, PGA alone was implanted into the other cornea.
The engineered corneal stroma became transparent gradually over a period of 8 weeks. The histology of engineered stromal lamellar was relatively similar to that of natural one, no significant differences were found in the diameter of cornea collagen fiber [(29.4 +/- 4.7) nm] in experimental rabbits compared with control [(28.5 +/- 3.5) nm], Student's t-test: P = 0.1316 > 0.05. TEM demonstrated that collagen fibrils deposited in engineered stroma had a similar diameter compared to that of normal counterpart. In addition, Western blot showed the positive expression of type I collagen in the collagen fibrils. In contrast, no new stroma tissue was formed when PGA alone implanted. A green colored stroma was observed when engineered with GFP-labeled cells under fluorescence light microscope.
The results demonstrate that nearly transparent corneal stroma can be obtained by the technique of cornea engineering.
通过组织工程技术重建角膜基质。
从新生兔角膜分离原代角膜基质细胞。当培养的细胞达到汇合状态时，将基质细胞与聚乙醇酸（PGA）混合以形成细胞-支架构建体。体外培养1周后，将构建体植入母兔角膜基质中。部分角膜基质细胞用绿色荧光蛋白（GFP）基因转染作为移植细胞的标记。8周时收获组织进行透射电子显微镜（TEM）、组织学和蛋白质印迹评估。对照组则将PGA单独植入另一角膜。
工程化角膜基质在8周内逐渐变得透明。工程化基质层板的组织学与天然基质层板相对相似，实验兔角膜胶原纤维直径[（29.4±4.7）nm]与对照组[（28.5±3.5）nm]相比，差异无统计学意义（Student's t检验：P = 0.1316＞0.05）。TEM显示工程化基质中沉积的胶原原纤维直径与正常对应物相似。此外，蛋白质印迹显示胶原原纤维中Ⅰ型胶原呈阳性表达。相比之下，单独植入PGA时未形成新的基质组织。在荧光显微镜下用GFP标记的细胞构建工程化角膜时可观察到绿色的基质。
结果表明，通过角膜工程技术可获得近乎透明的角膜基质。
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