以胶原凝胶为支架构建可植入工程化肝组织的实验研究

目的探讨肝细胞与液态Ⅰ型胶原复合构建可植入的工程化肝组织的可行性。方法SD大鼠肝细胞与液态Ⅰ型胶原及DMEM复合，形成肝细胞／胶原凝胶复合物。该复合物被接种在培养板中培养，采用相差显微镜和HE染色方法对培养肝细胞形态特征进行观察，并采用MTT法和免疫组化染色方法分别对肝细胞的活性和功能进行检测。肝细胞／胶原复合物同时被植入皮下腔中观察肝细胞的分化及工程化肝组织的形成情况，采用HE染色和免疫组织化学染色方法对植入的工程化肝组织进行评价。结果肝细胞与液态Ⅰ型胶原复合后形成凝胶状复合物，可见肝细胞均匀分布在整个复合物中，呈三维立体生长。在整个体外培养过程中，肝细胞始终保持圆形的形态；肝细胞在培养初期活性稍有下降，直至第7天时仍然保持活性的87％，之后随培养时间延长而活性逐渐降低。经过2周培养，肝细胞仍具有白蛋白的合成功能。皮下植入后1周，肝细胞／胶原复合物形成工程化灶性肝组织，免疫组化染色证实这种工程化的肝组织具有白蛋白的合成功能。结论采用胶原凝胶作为肝细胞生长和分化的支架可在体内构建类肝样的工程化肝组织。这种可植入的工程化肝组织提供了一种基于肝细胞治疗的新途径，并有望用于损伤肝组织修复和重建。

Experimental study of implantable engineered liver tissue using type I collagen gel as scaffold

OBJECTIVE: To construct implantable engineered liver tissue (ELT) using type I collagen gel as scaffold. METHODS: Type I collagen was obtained from the tail of a rat. Hepatocytes were collected from a Sprague-Dawley rat, mixed with liquid type I collagen and Dulbecco's modified Eagle's medium to create hepatocyte/collagen gel construct. The construct was inoculated in a 96-well plate. 0, 3, 5, 7, 9, 11, 13, and 15 days after the inoculation the viability of hepatocytes in vitro was measured by MTT assay. Phase contrast microscopy was used to observe the morphology of the hepatocyte/collagen gel construct. Three SD rats underwent injection of the hepatocyte/collagen gel construct into the subcutaneous space. One week later the implant was taken out. The morphology was conducted by routine H.E. staining and immunohistochemical staining. The morphology and function of hepatocytes was investigated by inverted microscopy, routine H.E. staining and immunohistochemical staining. The constructs were also implanted into subcutaneous space, and the differentiation of hepatocytes and the formation of engineered liver tissue were observed by routine H.E. staining and immunohistochemical staining. RESULTS: Phase contrast microscopy showed that the hepatocytes were distributed evenly in the construct and remained round-shape throughout the in vitro culture. MTT assay demonstrated that the high viability of hepatocytes (87%) was maintained up to 7 days, and then decreased gradually. Albumin, the specific marker of hepatocytes remained positive by immunohistochemical staining after 15-day culture. One week after implantation into subcutaneous space, the implanted hepatocytes retained its hepatocyte-specific morphology, i.e. round shape, large nuclear/cytoplasm ratio as well as binuclear cells, and formed small engineered liver tissue containing blood vessels within and surrounding the tissue. CONCLUSION: A novel approach to construct implantable engineered liver tissue using collagen gel as scaffold for growth and differentiation of hepatocytes has been dev eloped. This technique is an attractive tool for the development of liver tissue engineering.